Expression of calcium channels and related molecules.

<p>Relative gene expressions of (<b>A</b>) the calcium voltage-gated channel auxiliary subunit alpha2/delta1 (<i>Cacna2d1</i>); (<b>B</b>) the ryanodine receptor 2 (<i>Ryr2</i>); (<b>C</b>) the inositol 1,4,5-trisphosphate receptor type 1 (<i>Itrp1</i>); (<b>D</b>) the calcium release-activated calcium modulator-1 (Orai-1) and (<b>E</b>) the stromal interaction molecule 1 (<i>Stim1</i>) in pulmonary artery and thoracic aorta from control Wistar (CTRL; n TA = 14 rings; n PA = 14 rings; white bars) and spontaneously hypertensive rats (SHR; n TA = 12 rings; n PA = 16 rings; black bars). Results are expressed as means ± SEM. * 0.01</p

Role of the MAPKK and PI3K signaling in leptin induced response in thoracic aorta cells.

<p>Measurements of the intracellular calcium fluorescent signals [assessed by (Fc-Ff)/F0, where Fc corresponds to maximal cytosolic fluorescence during the last minute before the addition of the next drug, Ff corresponds to background fluorescence and F0 corresponds to mean fluorescence of the 6 last measurements before the addition of the first drug] in control thoracic aorta smooth muscle cells treated with (<b>A</b>) leptin (200 nmol/L) followed by the addition of a MAPKK inhibitor PD98059 (PD; 50 μmol/L; n = 35 cells), (<b>B</b>) MAPKK inhibitor PD98059 (PD; 50 μmol/L) followed by treatment with leptin (200 nmol/L; n = 33 cells), (<b>C</b>) leptin (200 nmol/L) followed by the addition of a PI3K inhibitor wortmaninn (wort; 10 μmol/L; n = 31 cells), and (<b>D</b>) PI3K inhibitor wortmaninn (wort; 10μmol/L) followed by treatment with leptin (200 nmol/L; n = 30 cells). Six rats were used for primary culture of vascular smooth muscle cells isolated by the explant technique from thoracic aorta and pulmonary artery. Results are expressed as means ± SEM. * 0.01</p

Measurement of cytosolic calcium concentration after leptin treatment in primary vascular smooth muscle cells.

<p>(<b>A</b>) Comparison of cytosolic calcium concentration after leptin treatment (200 nmol/L) in primary vascular smooth muscle cells [including thoracic aorta (TA) and pulmonary artery (PA) smooth muscle cells]] from control Wistar (CTRL) and spontaneously hypertensive rats (SHR) (n = 48 ± 12 cells). Six control Wistar rats and three SHR were used for primary culture of vascular smooth muscle cells isolated by the explant technique from thoracic aorta and pulmonary artery. Cytosolic calcium concentration was measured as the intracellular calcium fluorescent signals, assessed by (Fc-Ff)/F0, where Fc corresponds to maximal cytosolic fluorescence during the last minute before the addition of the next drug, Ff corresponds to background fluorescence and F0 corresponds to mean fluorescence of the 6 last measurements before the addition of the first drug. The base corresponds to mean fluorescence of the 6 last fluorescent measurements before the addition of the first drug. Results are expressed as means ± SEM.** 0.001<b><</b>p<0.01, ***p<0.001.Time course of the intracellular calcium fluorescent signal (assessed by fluorescence microscopy after loading with the Fluo-4 sensor) after leptin treatment (200 nmol/L) in smooth muscle cells from (B) control thoracic aorta, (C) SHR thoracic aorta, (D) control pulmonary artery, (E) SHR pulmonary artery.</p

Primers used for real-time quantitative polymerase chain reaction in <i>rattus norvegicus</i> thoracic aorta and pulmonary arteries.

<p>Primers used for real-time quantitative polymerase chain reaction in <i>rattus norvegicus</i> thoracic aorta and pulmonary arteries.</p

Concentration-response curves to leptin in endothelium-denuded rings from spontaneously hypertensive rats (SHR).

<p>Endothelium-denuded thoracic aorta and pulmonary artery rings were treated with a cumulative addition of leptin (1 pmol/L to 100 nmol/L) after preincubation with or without a MAPKK inhibitor PD98059 (PD; 50 μmol/L) (<b>A</b> and <b>B</b>) or a PI3K inhibitor wortmaninn (wort; 10 μmol/L) (<b>C</b> and <b>D</b>).Vasoactive responses were expressed as the percentages of the maximal tension response obtained with 80 mmol/L KCl. Thoracic aortic and pulmonary artery segments were collected in spontaneously hypertensive rats (n = 6 rings). Results are expressed as means ± SEM. * 0.01</p

Expression of transient receptor potential cation channels subfamily C (Trpc) members.

<p>Relative gene expressions of (<b>A</b>) <i>Trpc1</i>, (<b>B</b>) <i>Trpc3</i>, (<b>C</b>) <i>Trpc4</i> and (<b>D</b>) <i>Trpc6</i> in pulmonary artery and thoracic aorta from control Wistar (CTRL; n TA = 14 rings; n PA = 14 rings; white bars) and spontaneously hypertensive rats (SHR; n TA = 12 rings; n PA = 16 rings; black bars). Results are expressed as means ± SEM. * 0.01</p

Concentration-response curves to leptin in endothelium-denuded rings from spontaneously hypertensive rats (SHR).

<p>Endothelium-denuded thoracic aorta and pulmonary artery rings were treated with a cumulative addition of leptin (1 pmol/L to 100 nmol/L) after preincubation with or without (<b>A</b> and <b>B</b>) a calcium free medium with EGTA(1 μmol/L); (<b>C</b> and <b>D</b>) a L-type voltage-dependent calcium channel blocker nifedipine (Nif, 3 μmol/L); (<b>E</b> and <b>F)</b> a TRPC blocker SKF-96365 (30 μmol/L), or <b>(G</b> and <b>H)</b> caffeine (Caf) (30 mmol/L) + Thapsigargine (Tap) (100 nmol/L) + FCCP (100 nmol/L) allowing to empty intracellular calcium stores.Vasoactive responses were expressed as the percentages of the maximal tension response obtained with 80 mmol/L KCl. Thoracic aortic and pulmonary artery segments were collected in spontaneously hypertensive rats (n = 20 rings). Results are expressed as means ± SEM. * 0.01</p

Concentration-response curves to leptin in artery rings from hypertensive rats and normotensive rats.

<p>Endothelium-denuded (-) thoracic aortic (TA) and pulmonary artery (PA) rings from spontaneously hypertensive rats (SHR) and control Wistar rats (CTRL) were treated with a cumulative addition of leptin (1 pmol/L to 100 nmol/L). Vasoactive responses were expressed as the percentages of the maximal tension response obtained with 80 mmol/L KCl. Thoracic aortic and pulmonary artery segments were collected in control Wistar rats (n = 8 rings) and in spontaneously hypertensive rats (n = 14 rings). Results are expressed as means ± SEM. ** 0.001</p